Dendritic cells (DCs) play a pivotal role in the immune response, by collecting pathogens and tumor cell debris in peripheral tissues, and delivering these as peptides to lymph nodes for presentation to T cells. DC function relies heavily on the actin cytoskeleton, and mutations in actin-regulatory proteins like WASp lead to defects in DC function associated with immunodeficiency and autoimmunity. In T cells, we showed previously that WASp works together with another actin regulatory protein, HS1. HS1 is also expressed in dendritic cells, and our preliminary data show that HS1 is required for efficient antigen presentation. In addition, we find that HS1 is required for organization of podosomes, structures required for adhesion to the extracellular matrix and matrix metalloproteinase deposition during cell migration through tissues. We hypothesize that HS1 works together with WASp and dynamin II in DCs, to control receptor-mediated endocytosis and podosome- dependent migration to lymphoid organs. To test this hypothesis, we will carry out three Aims. First, we will test the role of HS1 interactions with dynamin II in driving vesicle internalization during receptor-mediated endocytosis, and ask if HS1 is also required for additional steps the intracellular transport and processing of internalized antigens. Second, we will investigate lamellipodial and podosome dynamics in migrating BMDCs using video microscopy, ask if HS1 is required for secretion of matrix metalloproteinases, and test the ability of HS1-/- DCs to migrate to lymph nodes in vivo. Third, we will conduct structure-function studies to test the hypothesis that the C-terminal SH3 domain of HS1 directs its actin-regulatory functions during endocytosis and migration via regulated interactions with WASp and dynamin II. Taken together, these studies will represent the first careful analysis of HS1 function in dendritic cells, and will provide important new insights into how actin regulatory pathways control specific aspects of DC function. This information will be valuable for developing therapeutic strategies for immunodeficiency and autoimmune diseases that arise as a result of mutations in actin-regulatory proteins, and for the development of effective DC-based vaccines and immunotherapies. PUBLIC HEALTH RELEVANCE: Several aspects of dendritic cell function depend on highly orchestrated changes in cell structure, and mutations in key cytoskeletal regulatory proteins lead to severe immunodeficiency or Lupus. This proposal addresses the role of one important cytoskeletal regulatory protein, HS1, in specific aspects of dendritic cell function. The knowledge gained from these studies will be important for understanding the basis of immunodeficiency and autoimmune disease, and for the development of effective dendritic cell-based vaccines.